The 14-O-acyl anthracyclines, represented by N-benzyladriamycin-14-valerate (AD 198) and N-benzyladriamycin-14-pivalate (AD 445), are functionally distinct from doxorubicin (DOX) and other DNA-binding anthracyclines. AD 198 and AD 445 localize in the cytoplasm, specifically bind to the C1 regulatory domain of protein kinase C (PKC) and activate PKC in a manner that rapidly triggers apoptosis in proliferating cells. As we have shown, AD 198 and AD 445 activate mitochondrial-associated PKC-d holoenzyme to trigger mitochondrial membrane depolarization and cytochrome c release in 32D.3 routine myeloid cells. Unlike non-PKC binding anthracyclines, such as DOX, AD 198 and AD 445 cytotoxicity is unaffected by Bcl-2 or Bcl-XL anti-apoptotic protein expression or p53 protein dysfunction in a variety of cell types. AD 198 and AD 445 also circumvent resistance conferred by multidrug transport proteins and altered topoisomerase II activity, In vivo, AD 198 demonstrates tumoricidal activity greater than DOX, with reduced myelosuppression and no significant organ toxicity, including a lack of cardiotoxicity. This proposal describes the next stages of analysis of AD 198 and AD 445 leading to clinical trials. The overall goals of this study are first, to analyze the mechanism by which PKC activation by AD 198 and AD 445 leads to rapid apoptosis. We will begin with 32D.3 cells to determine how drug-induced PKC-d activation leads to mitochondrial depolarization and cytochrome c release in a manner that is unaffected by Bcl-2 or Bcl-XL status. Second, given the clear advantage of these agents over DOX thus far in vitro and in vivo, we will continue these studies by assessing the in vivo efficacy of AD 198 an d AD 445 against implanted HL60 human leukemia cells expressing a variety of resistance mechanisms (Bcl-21P-gplMRPIp53-1-). Completion of these studies will establish in greater detail a mechanism of AD 198 and AD 445 induction of apoptosis and determine whether these novel agents can circumvent multiple, clinically relevant mechanisms of drug resistance in vivo.